1. Field of the Invention
The present invention relates to a process for preparing a human thrombin concentrate intended for therapeutic use, purified on an industrial scale from an isolated fraction of human plasma.
Thrombin is a serine-protease generated in the blood stream through activation of its inactive precursor, prothrombin. It plays a fundamental part in the coagulation process: it cuts the fibrinogen into fibrin monomers and, through proteolytic cutting, it activates Factor XIII which stabilizes the fibrin network.
It also plays a part in other physiological processes: it initiates the secretion and platelet aggregation reactions, thus facilitating the formation of the white plug. It also activates the proteins of the complement system. It has a mitogenic effect on the fibroblasts, which accelerates the healing of the damaged blood vessels.
As a result of these different properties, thrombin has therapeutic applications as a local haemostatic agent. At the present time, animal thrombin, equine or bovine, is used clinically in said therapeutic applications. These preparations are not always perfectly purified and their application can be the cause of immunological reactions due to the overload of heterologous proteins. The use of bovine thrombin further carries the risk of transmitting infectious diseases recently diagnosed and as et controlled with difficulty, such as spongiform bovine encephalitis (or "mad cow disease").
Thrombin purification, like that of many enzymes, poses problems as regards maintaining its total enzymatic activity. Native .alpha.-thrombin is, in fact, generally unstable and, through autolysis or limited proteolysis, it yields derivatives, .beta.- and .gamma.- thrombins that have lost a substantial part of their coagulating activity on fibrinogen.
Thrombin purification processes also have the drawback of being unpredictable with regard to scale-up from the laboratory or bench scale to an industrial scale. Given the large amounts of blood involved in industrial scale processes, this drawback is significant.
2. Description of Related Art
Known purification techniques apply especially to bovine thrombin: the include chromatography on ion exchange resins such as DEAE-cellulose (Yin et al. J. Biol. Chem., 1968, 243 112), possibly associated with filtration on SEPHADEX G 100.RTM. (Baughman et al. J. Biol. Chem., 1967, 242, 5252-5259); certain methods can be used to separate the .beta. and .gamma. forms on a phosphocellulose column (Rosenberg et al., J. Biol.Chem. 1970,245,5049)and through the association of several types of ion exchange chromatography (Batt et al. J. Biol. Chem. 1970, 245, 4857).
New resins have also been used, such as sulphoethyl or sulphopropyl SEPHADEX (Lundblad-Biochemistry, 1971, 10, 2501) as well as affinity chromatographies on supports such as heparin-SEPHAROSE (Nordeman et al. Thromb. Res., 1977, 11, 799-888) and p-aminobenzamidine-agarose (Hixson et al. Arch. Biochem. Biophys., 1973, 154,501).
Two publications describe the purification of human thrombin, one on benzamidine-SPHERODEX (Lorne et al. Rev. Fr. Transf. Hemobiol., 1989, 32, 391-403), but the product only exhibits low activity (11 to 20 NIH U/ml), and the other on new polystyrenes (Fischer et al. J. Chromatography, 1986, 363, 95-100), but this Product contains bovine Factor V (see below).
To obtain thrombin, it is necessary to have at one's disposal not only a source of its precursor, prothrombin, but also, depending on the mode of activation chosen, a sufficient concentration of the other coagulation factors involved in the activation process. Various methods have been described permitting the activation of prothrombin to produce thrombin. Fenton et al. (Biochim Biophys. Acta, 1971, 229, 26-32 and J. Biol. Chem., 1977, 252, 3587-3598) describe the preparation of thrombin from Cohn's fraction III extracted on resin, through the addition of calcium chloride and tissue thromboplastin extracted from the human brain. The reaction can be accelerated by the addition of Factor V of bovine origin (Bernamon-Djiane-Coagulation, 1968, 1, 259).
Specific activators extracted from snake venoms can also be used (Gosh et al. Thromb. Res., 1980, 20, 281).
However, these different methods present major drawbacks when one contemplates preparing very large volumes of human thrombin intended for therapeutic use. For instance, thromboplastin from the human brain is difficult to obtain and represents a limiting factor when it comes to treating several hundred liters of plasma. It is difficult to make activation with viper venom compatible with use in man, unless there is a high-performance system available to enable it to be eliminated subsequently. The use of bovine Factor V has proven particularly dangerous because the residual quantities that may be injected into patients are the origin of serious immunological reactions.